We thank Drs. Alison Heffer and Yong Lu for setting up the microinjection apparatus and sharing their invaluable knowledge and experience with insect RNAi. This work was supported by the National Institutes of Health (R01GM113230 to L.P.).

1. Rearing of D. maculatus
NOTE: A breeding colony of D. maculatus was set up in the authors&#39; lab using adults and larvae purchased commercially. The species identity was verified using DNA barcoding53.
<ol>
	<li>To set up a new cage in the lab, spread a thin layer of wood shavings into a medium-size insect cage (30.5 x 19 x 20.3 cm3). Place a ~ 10 x 6 x 3 cm3 chunk of Styrofoam in the cage to let larvae hide for pupation. Add 20 - 50 beetles (either adults or late instar larvae). Beetles will hide in the wood shavings.</li>
	<li>Add wet cat food in a Petri dish or a weighing boat. Cover the cage with mesh cloth and place the cage in an incubator.</li>
	<li>For maintaining a breeding colony in the lab, set the temperature between 25 and 30 &#176;C. D. maculatus grows faster at higher temperatures, but this also promotes the growth of fungus and mites. To maintain a healthy colony, use 25 - 28 &#176;C for regular stock maintenance and 30 &#176;C for rapidly expanding the colony. The life cycle takes approximately three weeks to four months depending on environmental factors50 (Figure 2).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54976/54976fig2.jpg" /> 
Figure 2: D. maculatus Lab Colony. Photograph of a typical D. maculatus insect cage is shown. Wood shavings are spread to let the beetles hide. Cat food is added in a small Petri dish or weighing boat. Styrofoam is placed into the cage as a refuge for final instar larvae to pupate. The cage shown here is 30.5 x 19 x 20.3 cm3 and houses a few hundred larvae, pupae, and adults. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Leave eggs in the cage to mature. To expand the colony, establish new cages with eggs (collected as described in Section 2), larvae, or adults, as above. D. maculatus lay eggs throughout the cage, especially near the food source.</li>
	<li>Replace wet cat food about twice a week. 
	NOTE: Due to the unpleasant odor of wet cat food, alternative food sources were also tested (see Discussion).</li>
</ol>
2. Embryo Collection
<ol>
	<li>To set up a collection, separate at least 50 males and 50 females from the colony. Young adults are best. Place adults in a mini-sized cage (17.8 x 10.2 x 12.7 cm3) without wood shavings. Add cat food in a weighing boat.</li>
	<li>Before starting collection, check the cage carefully for any embryos present and remove them. Put a stretched cotton ball into the cage, and leave the cage in a 30 &#176;C incubator. After the appropriate time window, the cotton ball will be ready for embryo collection.</li>
	<li>Fold a piece of black construction paper (A4 size) in half to create a crease, and then unfold.</li>
	<li>Remove the stretched cotton ball from the cage. Remove adults from the cotton ball and put them back into the cage.</li>
	<li>While pinching the stretched cotton ball very gently, tear it apart slowly into thin cotton filaments to let the eggs fall onto the black paper. 
	NOTE: D. maculatus embryos are fragile and hard to see in cotton. They can be easily crushed if holding the cotton ball too firmly.</li>
</ol>
3. dsRNA Preparation
<ol>
	<li>DNA Template Preparation for dsRNA Synthesis
	<ol>
		<li>Run 4 - 6 50 &#181;l PCR reactions using primers containing T7 promoter sequences at both 5&#39; ends to amplify DNA template according to manufacturer&#39;s protocol.</li>
		<li>Purify PCR product using a commercial PCR purification kit, following manufacturer&#39;s instructions. The ideal final concentration of DNA template is &#8805; 100 ng/&#181;l.</li>
	</ol>
	</li>
	<li>Injection Buffer Preparation
	<ol>
		<li>Prepare 100 ml of injection buffer (0.1 mM NaH2PO4, 5 mM KCl). Adjust pH to 6.8 with 5 M NaOH.</li>
		<li>Store aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>dsRNA Synthesis
	<ol>
		<li>Set up a reaction in a 0.2 ml PCR tube on ice and carry out an in vitro transcription reaction with T7 polymerase, following manufacturer&#39;s instructions, using ~ 500 ng template per reaction.</li>
		<li>Incubate the tube at 37 &#176;C overnight.</li>
		<li>Digest the DNA template with DNase, following manufacturer&#39;s instructions.</li>
		<li>Heat the tube to 94 &#176;C and hold at 94 &#176;C for 3 min.</li>
		<li>To anneal the dsRNA, slowly cool the tube by 1 &#176;C/min until it reaches 45 &#176;C after 1 hr. 
		NOTE: Steps 3.3.2 through 3.3.5 can be performed in a thermocycler.</li>
		<li>To ethanol precipitate&#160;of dsRNA, add 280 &#181;l of RNase free water to 20 &#181;l of reaction to adjust to a final volume of 300 &#181;l. Add 30 &#181;l of 3 M NaOAc (pH 5.2) and 650 &#181;l of ethanol. Place tube in -20 &#176;C freezer overnight.</li>
		<li>After centrifuging at 15,682 x g for 30 min at 4 &#176;C, wash the dsRNA pellet with 70% ethanol. Dry pellet and dissolve in 20 &#181;l injection buffer.</li>
		<li>Measure the concentration of the dsRNA using a spectrophotometer at A260. The ideal concentration is 3 - 5 &#181;g/&#181;l. Check the quality by running 5 &#181;l of a 1:25 dilution of dsRNA on a 1% agarose gel at 90 V for 30 min. A single band of expected size will be readily visible.</li>
		<li>Store the dsRNA at -20 &#176;C or colder. 
		NOTE: For every RNAi knockdown experiment, include a control dsRNA, which would not be expected to impact the process being studied. gfp dsRNA is an excellent control for most experiments. Prepare and inject the control dsRNA side-by-side with the experimental dsRNA.</li>
	</ol>
	</li>
</ol>
4. Assembly of Microinjector and Micromanipulator
NOTE: See Figure 3.
<ol>
	<li>Connect nitrogen or other air supply to the pressure input of a pneumatic pump instrument. If a pneumatic pump is not available, apply pressure using a 50 ml syringe connected to fine tubing.</li>
	<li>Connect a foot switch to the remote connector of the pump to control the eject pressure flow.</li>
	<li>Attach a glass capillary holder to the eject pressure port of the pump with tube.</li>
	<li>Fix the capillary holder on a micromanipulator close to a dissecting microscope.</li>
</ol>
5. Embryonic RNAi
NOTE: Figure 4 shows a flowchart of D. maculatus embryonic RNAi.
<ol>
	<li>eRNAi Preparation
	<ol>
		<li>Prepare an embryo collecting Petri dish lid (90 mm diameter). Place a piece of black filter paper to cover the inside of the lid. Put a standard microscope slide on the black paper.</li>
		<li>Dilute dsRNA to appropriate concentration with injection buffer. Use 2 - 3 &#181;g/&#181;l for initial experiments. Always keep dsRNA on ice prior to injection.</li>
		<li>Add food coloring at 1:40 dilution to the dsRNA and pipette gently several times to mix well.</li>
	</ol>
	</li>
	<li>Collecting Embryos for Injection 
	NOTE: At 25 &#176;C, nuclei in embryos 6 - 8 hr After Egg Laying (AEL) migrate towards the egg periphery53. A cellular blastoderm is established within 8 - 10 hr AEL53. To ensure embryos prior to cellular blastoderm stage are used for injection, 0 - 3 hr AEL embryos are recommended for use53. Embryo collection, preparation, and injection usually take less than 2 hr if the glass capillaries are in good shape. Therefore, the whole process can be completed within 5 hr AEL.
	<ol>
		<li>Collect embryos as described in steps 2.2-2.5.</li>
		<li>Tap the black construction paper to transfer embryos into collecting Petri dish lid.</li>
		<li>Stick double-sided tape along the edge of the slide.</li>
		<li>Using a paint brush, align the embryos on the tape perpendicular to the slide edge with the anterior or posterior end at the edge. Note that the anterior and posterior ends of early embryos are not readily distinguishable, thus on average half will be injected at the posterior end which is ideal.</li>
	</ol>
	</li>
	<li>Loading the dsRNA into the Glass Capillary
	<ol>
		<li>Take up 2 - 4 &#181;l of dsRNA into a 20 &#181;l microloader pipette tip.</li>
		<li>Insert the tip into a pre-pulled glass capillary (referred to as needle) and gently pipette the dsRNA solution into the capillary. Prepare needles from commercial glass capillaries using a micropipette puller. An example of an ideal pulled needle is shown in Figure 5. The length and taper of needles may need to be optimized after injection trials.</li>
		<li>Fix the glass capillary into the glass capillary holder.</li>
		<li>Assemble the capillary holder into the micromanipulator.</li>
	</ol>
	</li>
	<li>Injecting dsRNA into Embryos
	<ol>
		<li>Carefully transfer the slide with embryos onto the stage of the dissecting microscope.</li>
		<li>Move the slide to bring one embryo to the center of the field and focus microscope.</li>
		<li>Position the tip of the capillary with the micromanipulator while looking into the dissecting microscope. Bring the tip close to the end of the first embryo in the row.</li>
		<li>Switch on the nitrogen or other air supply.</li>
		<li>Set the solenoid input selector switch to vacuum.</li>
		<li>Adjust the eject pressure regulator to 10 - 15 psi. Adjust the pressure depending on the apparatus.</li>
		<li>Open the capillary if necessary. Once the tip is open, the colored dsRNA solution will fill the tip.</li>
		<li>Move the capillary tip forward to puncture the embryo. If the pressure setting is ideal, no embryonic fluid will flow into the capillary.</li>
		<li>Step on the foot switch to eject dsRNA solution into the embryo until an appropriate amount of solution is ejected. Figure 5 shows examples of embryo morphology after appropriate and inappropriate amounts of solution have been injected.</li>
		<li>Keep the capillary tip inside the embryo for ~ 2 sec, and then remove it.</li>
		<li>Move the capillary to the next embryo and repeat steps 5.4.8 - 5.4.10 until all embryos are injected. Change glass capillary if it gets clogged or breaks.</li>
	</ol>
	</li>
	<li>Post-injection Recovery and Incubation
	<ol>
		<li>After injection, place the slide in a Petri dish.</li>
		<li>Add a wet cotton ball to the Petri dish. Do not let the cotton ball touch the slide.</li>
		<li>Cover the Petri dish and wrap it with sealing film. Label the dish with the name of the dsRNA, concentration, date, time, and number of injected embryos.</li>
		<li>Place the Petri dish in a 30 &#176;C incubator until the embryos hatch.</li>
	</ol>
	</li>
</ol>
6. Parental RNAi
<ol>
	<li>Isolating Pupae for pRNAi
	<ol>
		<li>Collect pupae from the colony.</li>
		<li>Sort male and female pupae under microscope (see Figure 6). Keep them in two separate Petri dishes in a 30 &#176;C incubator to ensure that no mating occurs prior to injection.</li>
		<li>Check every day for eclosed adults. Pupation usually takes 5 - 7 days at 30 &#176;C.</li>
		<li>Transfer eclosed males and females (see Figure 6) to two separate Petri dishes and feed them cat food every other day until they are ready for injection.</li>
		<li>Proceed to injection once there are enough females and males at appropriate age. Females 4 to 8 days post-eclosion are best. For a typical analysis, 8 - 12 females are used. An equal number of males are needed for mating54.</li>
	</ol>
	</li>
	<li>Injecting dsRNA into Female Abdomen
	<ol>
		<li>Prepare dsRNA on ice (5.1.2 and 5.1.3).</li>
		<li>Attach a 32-gauge needle to a 10 &#181;l syringe.</li>
		<li>Anaesthetize females on CO2 stage.</li>
		<li>Load dsRNA solution into the syringe without taking up any air.</li>
		<li>Hold a female ventral side up with one hand and hold the syringe with the other.</li>
		<li>Gently penetrate the segmacoria (membrane) between sternites 2 and 3 with the tip of the needle. Figure 7 shows a female during injection. If the needle does not penetrate the tissue easily, angle the needle upward and slowly press down. Insert approximately 2 mm of the needle into the body.</li>
		<li>Check the syringe scale while slowly pushing the plunger, injecting ~ 2 &#181;l per female.</li>
		<li>After injecting, hold the needle still for at least 5 sec before removing it from the female&#39;s abdomen.</li>
		<li>Transfer the injected females to a Petri dish (90 mm diameter) and feed with cat food.</li>
		<li>Label the Petri dish with the dsRNA name, amount injected, date, and number of females.</li>
	</ol>
	</li>
	<li>Post-injection Recovery and Mating
	<ol>
		<li>Incubate the Petri dish in a 30 &#176;C incubator.</li>
		<li>After 24 hr, transfer injected females to a mini cage.</li>
		<li>Add to the cage an equal number of uninjected young males, and label the cage.</li>
		<li>Add a weighing boat with cat food into the cage.</li>
		<li>Leave the cage in a 30 &#176;C incubator to allow mating. After 24 hr, females should start to lay eggs and embryos can be collected daily or at required time intervals.</li>
	</ol>
	</li>
</ol>
7. Phenotypic Analysis after RNAi
NOTE: At 30 &#176;C, it takes ~ 55 hr for eggs to hatch50.
<ol>
	<li>eRNAi Viability Analysis
	<ol>
		<li>Calculate hatch rate by comparing the number of hatched larvae to the total number of injected embryos. Be sure to include a negative control injection as embryos may be harmed or killed by the injection procedure. Typical hatch rates vary from 30% - 60%. Beware of hatched larvae eating unhatched eggs.</li>
	</ol>
	</li>
	<li>pRNAi Viability Analysis 
	NOTE: If female viability is impacted by the gene targeted by RNAi, females injected with specific dsRNA but not negative control dsRNA will begin to die. If egg production or egg laying is impacted, females will exhibit partial or complete sterility. Score female survival compared to negative controls or compare total number of eggs laid by experimental and control females (7.2.3).
	<ol>
		<li>Set up embryo collection following step 2.2.</li>
		<li>After 24 hr, collect embryos following steps 2.4 - 2.5.</li>
		<li>Count the total number of the embryos.</li>
		<li>Transfer the embryos to a 90 mm Petri dish. Label the Petri dish with the gene targeted, collection date, and number of embryos.</li>
		<li>Incubate the embryos in a 30 &#176;C incubator until they hatch. Since hatched larvae feed on unhatched embryos, check the Petri dish frequently and separate hatched larvae from unhatched embryos using forceps (see Discussion).</li>
		<li>Count the number of hatched embryos and calculate the hatch rate.</li>
	</ol>
	</li>
	<li>Examining Cuticle Defects in Hatched Larvae
	<ol>
		<li>Collect hatched larvae in a 1.5 ml microcentrifuge tube.</li>
		<li>In a fume hood, add 1 ml of Fixation Solution.</li>
		<li>Leave the microcentrifuge tube at 4 &#176;C at least overnight to let the solution penetrate the larval tissue.</li>
		<li>To visualize, remove as much fixative from the tube as possible.</li>
		<li>Rinse larvae at least three times with PBST.</li>
		<li>Transfer larvae to a multi-well glass plate with PBST using a P-1000 pipette tip with the end cut off to widen it.</li>
		<li>Under a dissecting microscope, stretch larvae using forceps to examine defects. It is critical to stretch out larvae to examine defects as their bodies contract in fixative.</li>
	</ol>
	</li>
	<li>Examining Cuticle Defects in Unhatched Larvae 
	Note: This procedure is useful for examination of early embryonic phenotypes, especially for embryos that do not survive to hatching.
	<ol>
		<li>For pRNAi, add PBST into the Petri dish (7.2.4) and transfer unhatched embryos to a 1.5 ml microcentrifuge tube using a P-1000 pipette tip with the end cut off. For eRNAi, add PBST into the Petri dish (5.5.4), brush unhatched embryos off the microscope slide and transfer them to a microcentrifuge tube.</li>
		<li>Fix the embryos with Fixation Solution and rinse them with PBST for visualization following steps 7.3.2 - 7.3.6.</li>
		<li>Using forceps, dissect embryos out of the eggshell under a dissecting microscope in PBST.</li>
		<li>Transfer the embryos back to 1.5 ml microcentrifuge tube (~ 200 &#181;l of embryos in each tube).</li>
		<li>Remove as much solution as possible.</li>
		<li>Add 1 ml of 90% lactic acid/10% EtOH. Leave the centrifuge tube in a 60 &#176;C incubator at least overnight. 
		NOTE: Embryos can be left at 60 &#176;C for several days.</li>
		<li>To mount the embryos, pipette them onto a microscope slide using a P-1000 pipette tip with the end cut off.</li>
		<li>Manually position the embryos with forceps to an ideal orientation.</li>
		<li>Cover the embryos with a cover-slip and visualize under microscope using DIC.</li>
	</ol>
	</li>
	<li>Examining Cellular and Molecular Defects 
	NOTE: This procedure is useful for investigating the underlying causes of cuticle phenotypes or lethality that is not accompanied by cuticle defects.
	<ol>
		<li>For pRNAi, separate embryos from cotton balls at appropriate development stage and transfer them into an embryo collecting basket. The collection basket can be the same or similar to that used for Drosophila embryo collections.
		<ol>
			<li>Make the egg basket from a 25 ml plastic scintillation vial with the bottom of the vial removed, and a circle cut out of the lid (roughly 1 cm diameter). Place a circle of super fine mesh about 2.5 cm in diameter inside the lid, and then screw the scintillation vial on and use upside-down.</li>
			<li>As the mesh can be easily removed once the embryos are washed, immerse the mesh in a microcentrifuge tube with water to recover any embryos sticking to the mesh.</li>
		</ol>
		</li>
		<li>For eRNAi, at appropriate stage, add PBST to the Petri dish. Brush the embryos off the microscope slide and transfer them to an embryo collecting basket. 
		NOTE: Fixation, in situ hybridization, antibody staining, and nuclear staining protocols can be found in Xiang et al. 201553.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

While a small number of sophisticated model systems (mice, flies, worms) were developed during the 20th century, the 21st century has seen a wave of new animal systems being developed in labs throughout the world. These new systems allow scientists to address comparative, evolutionary questions that cannot be probed using only the &#39;standard&#39; model systems. This deployment of new models requires the rapid development of methods for lab culturing, gene identification, and functional approaches in new species. Here, procedures for rearing D. maculatus in the lab and step-by-step protocols for embryonic RNAi, parental RNAi, and phenotypic analysis in this beetle were presented. The goal is that these descriptions encourage others to use D. maculatus in their own experiments and to make use of the approaches presented here to develop additional new model species.
While D. maculatus lab colonies are incredibly easy to maintain, one limitation of rearing this species is the unpleasant odor of the wet cat food given to the beetles as their source of food. To avoid this, alternative foods such as whey/soy protein powder, cheese, ground dog food, and&#160;powdered milk, were tested&#160;with or without wet cotton to alter humidity. Ground dog food was the most effective alternative and can be used for regular colony feeding. Survival was excellent (&#62;90%) from egg to adult in cages reared on just dry dog food in 80% relative humidity, with or without wet cotton added. However, supplementing this diet with wet cat food when collecting eggs to increase fecundity may be necessary when collecting large numbers of eggs. If dog food is used, old food should be removed regularly, as conditioned dog food was found to inhibit egg laying54. Dog food kibbles (K.R., preliminary results), cork, wood, paper, and other materials can also be used as refuges71. Interestingly, biodegradation of Styrofoam using mealworm beetle larvae has been reported72. Therefore, this was tested for D. maculatus larvae. Young D. maculatus larvae survived until adulthood with only Styrofoam or asbestos-containing materials and wet cotton (data not shown) but whether they eat and digest those materials remains to be determined.
This report is the first detailed protocol for carrying out functional genetic studies in D. maculatus. The use of RNAi here represents an expansion of this technique to a new model system. Several specific observations are worth noting with respect to RNAi knockdown experiments in D. maculatus and other non-model species. First, to guarantee that RNAi will be effective in D. maculatus, the same strain of D. maculatus used here should be employed. There is evidence from Tribolium castaneum that different strains show variation in RNAi phenotypes24,73, and mutant phenotypes often show dependence on genetic background in model species74-76. Also, there is anecdotal evidence for strain-dependence in other protocols, including in situ hybridization. Second, appropriate timing of injection is critical for successful RNAi in D. maculatus. Somewhat counterintuitively, the segmacoria is most easily penetrated after the cuticle has completely sclerotized, at least two days after eclosion. Therefore, virgin females 4 - 8 days after eclosion should be used. Older females experience lower fecundity than newly eclosed females and thus are not appropriate for injection. Third, injected dsRNA may get pushed out by the inner pressure of the female abdomen. To minimize the possibility of losing a large amount of dsRNA after injection, hold the needle in the abdomen for ~ 5 sec after injection and then remove it slowly (6.2.8). Meanwhile, avoid pressing the female&#39;s abdomen during or shortly after injection. To circumvent biased results caused by this issue, inject at least 8 females for each dsRNA to get enough offspring (~ 200 embryos daily) for phenotypic analysis. Fourth, high egg yield is important for providing unbiased data for phenotypic analysis after injection. On average, each D. maculatus female produces approximately 35-55 embryos daily. Egg yield depends on population size, female age, humidity, food availability, temperature (data not shown), and other environmental factors54. Usually, females lay more at 30 &#176;C than 25 &#176;C. Fifth, unhatched embryos can be cannibalized by hatched larvae. As hatched larvae are usually wild-type-like or only mildly affected, while unhatched embryos are usually more severely affected, this habit of D. maculatus larvae can cause biased results in a quantitative phenotypic analysis. Therefore, removal of hatched larvae as early as possible is strongly recommended.
Our lab has focused on D. maculatus segmentation, although many aspects of this species&#8212;including physiology, ecology, and pest control&#8212;are of great interest. For studying embryogenesis and segmentation, a series of darkly pigmented stripes on the dorsal side of D. maculatus larvae can serve as a natural marker for abnormal development. Also, characteristic setae rooted on the pigmented stripes of larvae can be used as an indication of segments. These morphological features, together with the finding that mildly or moderately affected embryos can survive to hatching, are advantages of the D. maculatus model to study the mechanisms involved in patterning the basic body plan.
Finally, the importance of carrying out highly controlled experiments&#8212;including a negative control such as gfp dsRNA and testing two non-overlapping target regions for each gene&#8212;is critical to avoid misinterpretation due to effects of injection per se and&#160;off-target effects. For D. maculatus, 4 - 6 &#181;g (2 &#181;l of 2 or 3 &#181;g/&#181;l) dsRNA were injected into each female and the dsRNA was 200-250 bp long. Amounts and other details of the protocol may need to be optimized if targeting genes functioning later in development or in different physiological/metabolic processes. Previous discoveries showed that RNAi effects can be passed on to subsequent generations beyond the F1 generation in C. elegans15. A minority of hatched D. maculatus larvae with segmentation defects due to RNAi can survive until adulthood and can reproduce. Preliminary experiments failed to reveal obvious defects in the F2 generation. Future studies may reveal transgenerational effects of RNAi in this species.

1998 berichtete Fire and Mello , daß doppelsträngige RNA (dsRNA) Hemmung der Genfunktion in Caenorhabditis elegans 1 induzieren kann. Diese Antwort , ausgelöst durch dsRNA wurde RNA - Interferenz (RNAi) genannt, und solche RNAi-vermittelte Gen - Silencing wurde berichtet , in Tieren, Pflanzen konserviert zu sein, und Pilze 2-7. In Pflanzen und einige Tiere, systemisch RNAi - Funktionen, was bedeutet , dass der Effekt auf andere Zellen / Gewebe ausbreiten , wo wird dsRNA nicht direkt eingeführt ( zusammengefasst in 8-10). Wissenschaftler haben von dieser endogenen zellulären RNAi - Antwort von dsRNAs Gestaltung Gene von Interesse zu zielen, wodurch Klopfen nach unten Genfunktion , ohne direkt die Manipulation des Genoms ( zusammengefasst in 11-14) gemacht. RNAi ist ein leistungsfähiges Werkzeug für funktionelle Studien aufgrund der folgenden Vorteile. Erstens, selbst bei minimaler Gensequenz Informationen kann ein Gen gezielt RNAi verwendet. Dies ist besonders wichtig für studies von Nicht-Modellorganismen genomischen oder Transkriptom-Daten fehlen. Zweitens in Organismen, die RNAi-Antwort robust systemisch ist, kann RNAi-vermittelte Knock-Down bei fast jedem beliebigen Entwicklungsstadium durchgeführt werden. Diese Funktion ist sehr nützlich, um die Funktion von pleiotropen Gene zu studieren. Drittens, in einigen Fällen spreizen RNAi - Effekte auf die Gonaden und Nachkommen, so dass Phänotypen in Nachkommen 15,16 beobachtet werden. Dieses Phänomen, bekannt als elterliche RNAi (pRNAi), ist besonders vorteilhaft für Gene Embryonalentwicklung beeinflussen, zahlreiche Nachkommen von einem einzigen injiziert Mutter hergestellt ohne direkte Manipulation von Eiern untersucht werden. Aus diesen Gründen ist pRNAi die Methode der Wahl. Wenn jedoch pRNAi unwirksam ist, beispielsweise für die Gene für Oogenese erforderlich, dann embryonale RNAi (eRNAi) verwendet werden. Viertens kann RNAi verwendet werden, um das Äquivalent einer allelischen Serie, dass zur Erzeugung der Menge an dsRNA geliefert über einen Bereich variiert werden kann schwach bis stark Defekte zu produzieren. Eine solche Abstufung von Phänotypen können für das Verständnis der Genfunktion hilfreich sein, wenn das Gen in einem komplexen Prozess und / oder vollständigen Verlust der Funktion beteiligt ist, ist letal. Fünftens Lieferung von dsRNA ist in der Regel einfach und machbar, vor allem bei Tieren robusten Reaktionen systemischen RNAi zeigt. dsRNA kann durch Mikroinjektion 1,5, Fütterung / Aufnahme 17,18, Einweichen, 19,20 und Viren / Bakterien-vermittelte Abgabe 21,22 eingeführt werden. Sechstens, im Gegensatz zu einigen Gen-Targeting / Bearbeitungsmethoden, gibt es keine Notwendigkeit für Organismen tragen die Mutation zu screenen oder genetische Kreuzungen durchzuführen Homozygoten zu erzeugen, wenn RNAi verwendet wird. Daher kann im Vergleich zu vielen anderen Techniken zur Untersuchung der Genfunktion ist RNAi schnell, kostengünstig und kann für großflächige Bildschirme 23-25 aufgebracht werden. Die breite Anwendung von RNAi stellt Mittel bereit, funktionelle Studien in einem breiten Spektrum von Organismen durchzuführen, um den Bereich der Arten für das Studium zur Verfügung expandierenden beyond die traditionellen Modellsysteme für die genetische Werkzeuge entwickelt worden. Zum Beispiel können nicht-Modellsysteme Studien mit benötigt Einblicke in die Evolution von Genen und Gen - Netzwerken zu geben , indem die Funktionen von Orthologen von Spezies zu vergleichen , die unterschiedliche Entwicklung Modi oder ausstellenden verschiedene morphologische Merkmale 26-29. Diese Art von Studien wird ein besseres Verständnis der biologischen Vielfalt zur Verfügung stellen, mit Auswirkungen sowohl für angewandte und Grundlagenforschung. Als die größte Tiergruppe auf dem Planeten, Insekten bieten eine große Chance, die zugrunde liegenden Mechanismen Vielfalt zu erkunden. Darüber hinaus sind Insekten im Allgemeinen klein, haben kurze Lebenszyklen, hohe Fruchtbarkeit und sind leicht nach hinten im Labor. In den vergangenen zwei Jahrzehnten hat sich RNAi erfolgreich bei Insekten Spanning Aufträge angewendet worden, einschließlich Diptera (Zweiflügler) 5, Lepidoptera (Schmetterlinge und Motten) 30, Coleoptera (Käfer) 16,31, Hautflügler (SAWFLügen, Wespen, Ameisen und Bienen) 32, Hemiptera (true Bugs), Isoptera (Termiten) 34, Blattodea (Schaben) 35, Orthoptera (Grillen, Heuschrecken, Heuschrecken und katydids) 36 und Phthiraptera (Läuse) 37. Die erfolgreiche Anwendung von RNAi hat sich für die Untersuchung der Musterung in der frühen Embryonalentwicklung (anterior-posteriore Achse 32, dorsalen-ventralen Achse 28, Segmentierung 26,38) funktionelle Daten zur Verfügung gestellt, die Geschlechtsbestimmung 39,40, Chitin / Kutikula Biosynthese 41, Ecdyson 42 signalisiert, Sozialverhalten 43 und mehr. RNAi Methoden für verschiedene Insektenarten entwickelt werden , können von zusätzlichen Vorteil sein, dass sie geeignet sind , für die Schädlingsbekämpfung , nützlich zu sein ( zusammengefasst in 44-46). RNAi-Effekte werden genspezifische sowie artspezifischen, solange nicht konservierten Regionen für die Ausrichtung gewählt. Für nützlichen Insektenarten wie Honigbienen und Seidenwürmer, Gene von entscheidender Bedeutung für das Überleben der TargetingViren oder Parasiten - Infektion zu kontrollieren kann eine neue Strategie bieten diese Spezies 47,48 zu schützen. Dermestes maculatus (D. maculatus), gebräuchliche Bezeichnung verbergen Käfer, ist weltweit mit Ausnahme der Antarktis verteilt. Als holometabolous Insekt umfasst das D. maculatus Lebenszyklus embryonale, Larven, Puppen und adulten Stadien (Abbildung 1). Weil es auf Fleisch ernährt, ist D. maculatus in Museen verwendet tote Tiere zu skeletonize und forensische Entomologen können es verwenden Zeitpunkt des Todes 49,50 abzuschätzen. D. maculatus ernährt sich von tierischen Erzeugnissen einschließlich Kadaver, getrocknetes Fleisch, Käse, und die Puppen / Kokons von anderen Insekten und somit verursacht Schäden an Haushalte, Vorrats und die Seide, Käse und Fleischindustrie 51,52. RNAi in diesem Käfer Anwendung könnte eine effiziente und umweltfreundliche Art und Weise zur Verfügung stellen ihre wirtschaftlichen Auswirkungen zu minimieren. Unser Labor hat D. maculatus als neues m verwendetodel Insekt zu studieren Segmentierung 53. Neben den Lab - Aufzucht zugänglich ist, ist D. maculatus von Interesse für die Grundlagenforschung , wie es ein Zwischenkeim - Entwickler ist es eine nützliche Art den Übergang zwischen kurz- und langfrisKeim Entwicklung zu studieren. <img alt="Abbildung 1" src="/files/ftp_upload/54976/54976fig1.jpg" /> Abbildung 1: Lebenszyklus von D. maculatus. Fotografien von D. maculatus in verschiedenen Lebensphasen, wie angegeben. Der Lebenszyklus vom Ei bis zum erwachsenen dauert drei Wochen bei 30 ° C, jedoch bei niedrigeren Temperaturen länger. (A, F) frisch gelegten Embryonen sind weiß bis hellgelb und oval, etwa 1,5 mm in der Länge. Embryonalentwicklung dauert ca. 55 Stunden bei 30 ° C. (B, C und G) Larven haben dunkle pigmentierte Streifen und sind mit Setae bedeckt. gehen Larvae durch mehrere instars abhängig von der Umgebung und deren Länge bis über 1 cm verlängern kann. (D, H) </strOng&gt; Junge Puppen sind hellgelb. Verpuppung erfolgt ~ 5 bis 7 Tage bei 30 ° C. (E, I) Kurz nach dem Schlüpfen, erscheint dunkle Pigmentierung über die erwachsenen Käfer Körper. Erwachsene können bis zu mehrere Monate leben und eine Frau Hunderte von Embryonen über ihr Leben legen. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Bisher haben wir gezeigt , dass RNAi in Klopfen nach unten Genfunktion in D. wirksam ist maculatus 53. Hier unsere Erfahrung Aufzucht D. maculatus Kolonien im Labor wird zusammen mit Schritt- für -Schritt - Protokolle geteilt sowohl für die embryonale und die elterliche RNAi Set-up, Injektion, Nacheinspritzung Pflege und phänotypische Analyse. Die dsRNA-vermittelte Knock-Down und Analyseverfahren hier eingeführt nicht nur detaillierte Informationen zu Fragen in D. maculatus Adressierung, sondern auch potenzielle Bedeutung haben for RNAi in anderen nicht-Modell Käfer / Insektenarten anwenden.

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dermestes maculatus live beetles</td>
			<td style="width:140px;">Our lab or Carolina Biological Supply</td>
			<td style="width:119px;">#144168</td>
			<td style="width:167px;">Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wet cat food</td>
			<td style="width:140px;">Fancy Feast</td>
			<td style="width:119px;"></td>
			<td>Chunks of meat with gravy. Can buy at most pet food and grocery stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry dog food</td>
			<td style="width:140px;">Purina Puppy Chow</td>
			<td style="width:119px;"></td>
			<td>Can buy at most pet food and grocery stores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect cage (size medium, 30.5x19x20.3 cm)</td>
			<td style="width:140px;">Exo Terra</td>
			<td>PT2260</td>
			<td>For colony maintenance. Can use larger cage if needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect cage (size mini, 17.8x10.2x12.7 cm)</td>
			<td style="width:140px;">Exo Terra</td>
			<td>PT2250</td>
			<td>For embryo collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">89038-968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cotton ball</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">22-456-883</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Megascript T7 transcription kit</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">AM1334</td>
			<td>For 40 reactions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pneumatic pump</td>
			<td style="width:140px;">WPI</td>
			<td style="width:119px;">PV830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Capillary holder</td>
			<td style="width:140px;">WPI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:140px;">NARISHIGE</td>
			<td style="width:119px;">MN-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black filter paper (90 mm)</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">28342-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Food coloring (green)</td>
			<td>McCormick</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Borosilicate glass capillary</td>
			<td style="width:140px;">Hilgenberg</td>
			<td style="width:119px;">1406119</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle puller (micropipette puller)</td>
			<td style="width:140px;">Sutter Instrument Co.</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope glass slide</td>
			<td>WorldWide Life Sciences Division</td>
			<td style="width:119px;">41351157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sealing film (Parafilm M)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">13-374-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Model 801 Syringe (10 &#181;l )</td>
			<td style="width:140px;">Hamilton</td>
			<td style="width:119px;">7642-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle (32-gauge)&#160;</td>
			<td style="width:140px;">Hamilton</td>
			<td style="width:119px;">7762-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fixation Solution (Pampel&#39;s)</td>
			<td>BioQuip Products, Inc.</td>
			<td style="width:119px;">1184C</td>
			<td>Toxic, needs to be handled in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep (DUMONT #5)</td>
			<td style="width:140px;">Fine Science Tools</td>
			<td style="width:119px;">11252-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover slip (24X50 mm, No. 1.5)</td>
			<td style="width:140px;">Globe Scientific</td>
			<td style="width:119px;">1415-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Femtotips Microloader pipette tip</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">E5242956003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscopy for embryo injection</td>
			<td style="width:140px;">Leica</td>
			<td style="width:119px;">M420</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscopy for larval phenotypic visualization</td>
			<td style="width:140px;">Zeiss</td>
			<td colspan="2">SteREO Discover. V12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DIC microscopy</td>
			<td style="width:140px;">Zeiss</td>
			<td style="width:119px;">AXIO Imager. M1</td>
			<td></td>
		</tr>
	</tbody>
</table>

rearing and double-stranded rna-mediated gene knockdown in the hide beetle, dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

The authors&#39; lab has used RNAi technology to study the functional evolution of genes regulating segmentation in insects53,55. While all insects are segmented, the genes regulating this process appear to have diverged during insect radiations26,38,56-63. Genetic screens in Drosophila identified a set of nine pair-rule segmentation genes that are responsible for promoting the formation of body segments64-70. Here, the ortholog of one of these genes, paired (prd), is used to document the utility of RNAi for studying gene function in D. maculatus.
eRNAi and pRNAi were each effective in demonstrating roles for Dmac-prd in segment formation in this species. 2 - 3 &#181;g/&#181;l of dsRNA designed to target Dmac-prd (Figures 8B, 8D and 8F) was compared to gfp dsRNA, injected as negative control (Figures 8A, 8C and 8E).
Control offspring hatched with one pigmented stripe per segment. Neighboring pigmented stripes were separated by a non-pigmented gap (Figure 8A). After prd pRNAi, affected offspring hatched with fused neighboring pigmented stripes, indicating segmental boundaries were defective (Figure 8B). Depending on phenotypic severity, one or several fusions were detected in affected larvae. Nevertheless, fusions consistently appeared at the boundary regions between T2/T3, A1/A2, A3/A4, A5/A6, A7/A8, indicating pair-rule-like defects. Cuticle phenotypes after prd knockdown showed loss of abdominal segments as well as shortened body length (Figure 8D). Engrailed (En) antibody staining was performed to examine molecular defects in early embryos after prd knockdown. While control embryos showed striped En expression with equal intensity in every segment (Figure 8E), reduced En expression was detected in alternate stripes in offspring from Dmac-prd dsRNA injected females (asterisks in Figure 8F). The pattern of reduced En expression is consistent with the defective cuticle pattern observed in affected hatched larvae. For more detailed results of phenotypes after prd knockdown in D. maculatus, see Xiang et al. 201553.
<img alt="Figure 3" src="/files/ftp_upload/54976/54976fig3.jpg" /> 
Figure 3: Injection Apparatus. Photograph of the dissection microscope and micromanipulator used to inject D. maculatus embryos is shown. Nitrogen supply is connected to the pressure input port of a pneumatic pump. Glass capillary holder is connected to the eject pressure port of the pump. Foot switch is connected to the pump. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54976/54976fig4.jpg" /> 
Figure 4: A Flowchart for D. maculatus Embryonic RNAi. (A-D) Collecting embryos for injection. Females lay embryos (orange) in cotton balls (grey circle). Separate embryos from cotton balls and let them drop onto a piece of black construction paper. White bars indicate tears in cotton ball. Transfer embryos to a collecting Petri dish lid, lined with black paper. (E, F) Injecting dsRNA into embryos. Align embryos on slides on double stick tape and inject them individually under a dissecting microscope. Needle with green food dye is shown. (G-J) Post-injection recovery and incubation. Place a wet cotton ball in the Petri dish to provide humidity. Cover the Petri dish and wrap it with sealing film (light blue). Place the Petri dish in an incubator and remove hatched larvae for phenotypic analysis. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54976/54976fig5.jpg" /> 
Figure 5: Good vs. Bad Embryonic Injection Examples. (A) Uninjected embryo. (B) Embryo injected with too little dsRNA. (C) Embryo injected with appropriate amount of dsRNA. (D) Broken embryo with overflowing dsRNA caused by over-injection. Food coloring was added to dsRNA for visualization. (E) Examples of pulled capillary tubes used as needles for injection. Note the taper and tip length for two examples of functional needles. Scale bars for A-D represent 200 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54976/54976fig6.jpg" /> 
Figure 6: Male and Female Adults and Pupae. (A) Ventral view of a male pupa. (A&#39;) Magnification of posterior part of A. (A&#34;) Illustration of male pupa genitalia. (B) Ventral view of a female pupa. (B&#39;) Magnification of posterior part of B. Female pupae have two genital papillae (white arrows). (B&#34;) Illustration of female pupa genitalia. (C) Ventral view of a male adult. (C&#39;) Magnified view of posterior part of C. Note that male adults have trident-like genitalia and a circular lobe-like structure on the 4th sternite (white and black arrows, respectively). (D) Ventral view of a female adult. (D&#39;) Magnified view of posterior part of D. For all panels, scale bars indicate 1 mm. For details, see Xiang et al. 201553. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54976/54976fig7.jpg" /> 
Figure 7: A Female during Injection. Females are anesthetized and placed ventral side up. A needle penetrating the segmacoria between the 2nd and 3rd sternites to inject dsRNA is shown. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54976/54976fig8.jpg" /> 
Figure 8: Representative Phenotypes after Dmac-prd pRNAi. (A) Control injection. Lateral view of a hatched wild type-like gfp pRNAi offspring with three thoracic segments and ten abdominal segments. Each segment has setae and pigmented stripes. (B) Lateral view of a hatched prd pRNAi offspring. The gap between labeled neighboring pigmented stripes is narrowed or completely missing. (C) Cuticle phenotype of an unhatched control wild type-like embryo. (D) Cuticle phenotype of an unhatched prd pRNAi offspring with fewer abdominal segments. (E,F) Dissected germband from: (E) Control, gfp pRNAi has evenly expressed En in every segment, (F) prd pRNAi, En expression is reduced in alternate segments (asterisks). For all panels, scale bars indicate 500 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54976/54976fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus at JoVE.com

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Zucht- und RNA-vermittelte Gene Knockdown in der ausblenden Doppelstrang-Käfer,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

rearing-double-stranded-rna-mediated-gene-knockdown-hide-beetle

Nanyang Technological University

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

10.5K Aufrufe.  University of Maryland. Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Serie: Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Parallel Messung von Circadian Clock Genexpression und Hormon-Sekretion in primären humanen Zellkulturen

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Forschung

Genetik

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Herstellung von rAAV9 überexprimiert oder Sturz Gene in Mäuseherzen

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Das Studium der Zellzyklus-regulierten Genexpression durch zwei komplementäre Zellsynchronisationsprotokolle

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Eine Standardmethodik, vor-Ort-Mutagenität als Funktion der Punktmutation Reparatur katalysiert durch CRISPR/Cas9 und SsODN in den menschlichen Zellen zu untersuchen

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

Sequenz-spezifische und gezielte Anerkennung der Doppelstrang-RNA über Single-Stranded RNA durch chemisch modifiziert Peptid-Nukleinsäuren

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Effiziente Produktion und Kennzeichnung der CRISPR/Cas9 generiert gen Knockouts im Modellsystem Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Herstellung und Reinigung des Baculovirus für Gentherapie Anwendung

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

Eine Methode, um den C924T-Polymorphismus von Thromboxan-A2-Rezeptor-Gens zu studieren

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Die Wurzel Mikrobiom zu erkunden: Extrahieren von Bakteriengemeinschaft Daten aus dem Boden, die Rhizosphäre und die Wurzel Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Charakterisierung von Histon Post-translationale Modifikation Veränderungen in Hefe Neurodegenerative Proteinopathy Modelle

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...